Specific detection of proteolytic enzymes

ABSTRACT

Specific detection of proteolytic enzymes is achieved by extinguishing dye fluorescence by the amino acid tryptophan. Ttyptophan is disposed on one side of the cutting site of a proteolytic enzyme while an amino acid marked with a dye is arranged on the other side. Extinction of fluorescence occurs prior to enzyme cutting. Spatial separation of the tryptophan and the dye takes place after cutting, whereby fluorescence extinction does not occur. The dye can then fluoresce and a signal increase occurs thereby indicating that cutting has been carried out and the presence of the enzyme.

BACKGROUND OF THE INVENTION

Proteolytic enzymes are proteases or peptidases, i.e. enzymes that areable to split or lyse peptides or proteins. The word “peptide” generallydenotes a shorter amino acid sequence, whereas the word “protein”generally denotes larger molecules consisting of an amino acid sequence.Both terms are used synonymously in the following.

Peptidases and proteases play a decisive role in protein activation,cell regulation and signal transmission. Their detection at maximumpossible sensitivity is therefore of enormous importance forunderstanding the functioning of a cell and its communication, and formonitoring the course of diseases.

Furthermore, precise knowledge of peptidase or protease concentrationpermits the targeted development of new therapeutic agents forinhibition, e.g. of HIV protease. HIV protease breaks down the HIVprotein, which is at first very long after multiplication in a cell,into functioning individual proteins, which only then permit the furtheractivity and growth of the HIV virus. There are two possible approaches.Firstly, if the HIV protease is blocked, the HIV virus cannot replicateand its propagation is halted. Secondly, infection with the HIV viruscan take place via detection of the HIV protease. Specific detection ofHIV protease can therefore serve as an AIDS test.

Moreover, protease tests are attracting increasing attention in medicalresearch, because more and more diseases, including cancers, are beingconnected with enzymes. Tumor-associated proteases are increasingly theobject of oncological-biochemical research. These proteases bring aboutthe breakdown of proteins of the tumor stroma and of the basal membraneand thus permit tumor cell invasion. Therefore investigations ofprotease systems that are overexpressed in tumor cells are among the newprognosis factors in tumor diagnostics.

Another important aspect is the use of protease tests in quality controlof biochemical products. If, for example proteins, enzymes, peptides,etc. are being sold, it is necessary to ensure that the products soldare free from proteases, so that the product sold does not break down onits own. The same can be used for testing the specific activity ofproteases, e.g. after prolonged storage, regarding the question as towhether the enzymes are still active.

The proteolytic enzymes are divided into endopeptidases (proteases) andexopeptidases. Endopeptidases cleave amino linkages inside peptides.Exopeptidases digest peptides amino acid by amino acid starting fromtheir ends, i.e. they cleave terminal amino acids. The exopeptidases canbe further subdivided into aminopeptidases and carboxypeptidases, asclaimed in their activity at the amino or N end or at the carboxy or Cend of the peptide.

For determination of the concentration or activity of various peptidasesand proteases, to date various fluorescence-based tests have beendeveloped, and their operating principles and sensitivities are brieflydescribed below.

One possibility for detecting proteolytic enzymes is offered by the“ENZCHEK® Protease Assay Kit” from the company Molecular Probes (Eugene,USA). For detecting proteases, this kit employs casein derivatives, towhich very many pH-insensitive green or red fluorescing BODIPY® dyes arecoupled (Karolin J., Johansson B. A., Strandberg L., Ny T.; J. Am. Chem.Soc., 1994, 116, 7801-7806). On account of the very high degree ofmarking of the protein, the intermolecular distances between thefluorophores are very small (typically of the order of a fewnanometers). The dyes therefore extinguish each other, inter ella bydimer formation. Casein is a large enzyme, which contains many bindingsites for proteases, because it has many different sequence segments. Ifthe dye-marked casein derivative comes into contact with proteases, e.g.with trypsin, which hydrolyzes peptide bonds of the basic amino acidsarginine and lysine on the carboxy side, these split the caseinderivative into smaller peptides, so that as a rule the distance betweenthe dyes increases. Extinction does not occur, and an increase influorescence is observed, indicating that proteolytic enzymes arepresent. For trypsin, for example, the detection sensitivity of this kitis a few ng/ml. For these small quantities of protease the detectiontime or measurement time is more than 10 hours. This test detects manyproteolytic enzymes nonspecifically, since casein is digested byelastase, pepsin, thermolysin, papain, trypsin, etc.

An enzyme test based on Rhodamine 110 for various proteases andpeptidases (Leytus S. P., Melhado L. L., Mangel W. F.: Rhodamine-basedcompounds as fluorogenic substrates for serine protease, (1983) Biochem.J., 209 (2): 299-307; Leytus S. P., Patterson W. L., Mangel W. F.: Newclass of sensitive and selective fluorogenic substrates for serineproteinases. Amino acid and dipeptide derivatives of rhodamine, (1983)Biochem. J., 215 (2): 253-260) is also being marketed for the detectionof serine proteases. In this test, at least one of the amino groups ofRhodamine 110 is coupled covalently to an amino acid (usually arginine),causing strong extinction of the fluorescence of the fluorophore. Afterdigestion of the peptide bonds there is a dramatic increase influorescence of Rhodamine 110. For caspase-3, the sensitivity of thetest is a few ng absolute quantity of enzyme. Disadvantages are that theprinciple is not of general applicability, sometimes its action isnonspecific, and it is relatively insensitive.

A protease or peptidase test can also be accomplished by means ofForster fluorescence resonance energy transfer (FRET) (Forster Th.:Zwischenmolekulare Energiewanderung und Fluoreszenz [IntermolecularEnergy Transfer and Fluorescence], (1948) Annalen der Physik, 2: 55-75).In this, the specific identification sequence of an endopeptidase ismarked covalently at both ends with a donor dye and an acceptor dye.Owing to spectral overlap of donor emission with acceptor absorption,the fluorescence of the donor is extinguished at small distances. If thepeptide is cleaved by the endopeptidase, the spatially close contactbetween donor and acceptor is lost, leading to an increase influorescence. Detection of HIV protease will be briefly explained here,as an example. The specific cleaving sequence of HIV protease iscoupled, near the cleavage site, with an

The peptide sequence is then:Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys-(DABCYL)-Arg SEQ IDNO: 1, using the 3-letter abbreviations for the amino acids. Aftercleavage by the HIV protease we have two parts:Arg-Glu(EDANS)Ser-Gln-Asn-Tyr-OH SEQ ID NO: 2 andPro-Ile-Val-Gln-Lys(DABCYL)-Arg SEQ ID NO: 3. Suitable small distancebetween the two dyes. Sensitivity is of the order of nanomolar solutionsof HIV protease. A disadvantage is the expensive synthesis of thesubstrate with specific coupling of a donor and an acceptor. If thesubstrate is to some extent marked incompletely, e.g. with just onedonor, this leads to poor sensitivity of the test.

The aim of the invention is to improve the possibilities for a specificdetection of proteolytic enzymes.

This aim is achieved by the inventions as claimed in the independentclaims. Advantageous developments are described in the subclaims.

SUMMARY OF THE INVENTION

A first method of detection (“test” for short) is used for the detectionof endopeptidases (also briefly referred to as enzymes hereinafter) bymeans of special peptides.

For this purpose, a peptide is prepared with the properties enumeratedbelow. The peptide contains an identification sequence of theendopeptidase that is to be detected, and can therefore serve assubstrate for the enzyme, with a cleavage site between two amino acidsin the identification sequence in the peptide.

The peptide contains at least one quencher and at least one fluorophore.“Quenching” means reduction of the emission of the fluorophore due tointeraction with the aforesaid quencher. A fluorescent dye is used asthe fluorophore. Quenching causes a decrease in fluorescence quantumyield. The words “extinction” and “extinguishing molecule” or“extinguisher” are also used as synonyms for “quenching” and “quencher”.

The amino acid tryptophan is used as quencher. Other amino acids, suchas tyrosine or histidine, also exhibit slight fluorescence extinction,but do not have the strong quenching action of tryptophan.

The fluorescent dye is selected on the basis that its fluorescence canbe extinguished by tryptophan. Furthermore, it should be possible forthe fluorescent dye to be coupled covalently to peptides or proteins. Asa rule the fluorophore is coupled to an amino acid.

Attainment of high sensitivity of the test requires targeted selectionof the peptide sequence: the quencher must be arranged on one side ofthe cleavage site, with the fluorophore on the other side of thecleavage site. The sequence must be selected so that the quencher andthe fluorophore are sufficiently spatially close to one another, so longas the substrate has still not been cleaved by the enzyme at thecleavage site between quencher and fluorophore, to provide thepossibility of at least partial extinction of the emission of thefluorophore. After cleavage of the substrate by the enzyme at thecleavage site between quencher and fluorophore, a definite increase inemission of the fluorophore must be possible.

Thus, the peptide or protein is not marked with the fluorescent dye onthe tryptophan itself, but on another amino acid, which is separatedfrom the tryptophan by the cleavage site. Furthermore, an amino acidother than tryptophan is chosen for coupling the fluorescent dye, e.g.lysine, arginine or cysteine. The fluorescent dye can also be coupled toone end of the peptide or protein.

The largest increase in fluorescence is obtained if the tryptophan andthe dye-marked amino acid are located directly on either side of thecleavage site. For dye MR 121, for example, an increase in fluorescenceby a factor of 10 can be observed after cleavage of the peptide by theenzyme.

Moreover, no tryptophan is to be arranged in adjacent positions on thesame side of the cleavage site of the peptide sequence as the dye, andespecially not in immediately adjacent positions, so that the dye is notquenched by this additional tryptophan.

Appropriate choice of substrate sequence makes it possible to mark thesubstrate with just one fluorescent dye, and not with at least two, aswas necessary until now. This simplifies the synthesis of the substrate.In addition there is less disturbance of binding of the enzyme to thesubstrate by coupled dyes, resulting in greater specificity of bindingbetween substrate and enzyme.

Finally, the peptide thus prepared and the enzyme to be detected aremixed in a solution. If binding occurs between peptide and peptidase,the peptide is cleaved at the cleavage site. As a result, the spatialdistance between the fluorophore and the quencher becomes greater, andthe emission of the fluorophore increases. This increase in emission isdetermined and serves for detecting those peptidases or proteases thatbind to the identification sequence and can cleave the peptide there.Accordingly, the detection reaction is specific. It can be directedtowards the detection of specific enzymes by varying the peptidesequence.

In particular, the test can detect enzymes specifically, even when othertypes of enzymes are also present in the solution. For example, theenzyme trypsin can be detected in a solution that also contains elastaseand chymotrypsin.

If the temporal course of the increase in emission is traced, theconcentration of the enzyme can be determined quantitatively. Using thetest as claimed in the invention, it is even possible to detectproteases or peptidases at a concentration of 10^−15 mol/l.

As dyes, consideration may be given to all fluorescent dyes that aremeasurably extinguished in their fluorescence by the amino acidtryptophan. Dyes in the classes of oxazine derivatives (e.g. MR 121, JA242, JA 243) and rhodamines (e.g. JA 165, JA 167, JA 169), which absorband emit in the red region of the spectrum of visible light [see, forexample, Müller R., Zander C., Sauer M., Deimel M., Ko D. S., SiebertS., Arden-Jacob J., Deltau G., Marx N. J., Drexhage K. H., Wolfrum J.Chem. Phys. Let. 1996, 262, 716-722] are particularly suitable. Thesered dyes are extinguished by tryptophan by one to two orders ofmagnitude better than other dyes.

When red dyes are used there is a marked increase in sensitivity,because otherwise the autofluorescence of biologically relevantproteins, e.g. blood sera and ascites, which mostly occurs in the greenregion of the visible spectrum, makes sensitive detection moredifficult. With the method as claimed in the invention, sensitive testscan also be carried out on these biologically relevant samples, even onundiluted blood sera.

The aim is also achieved by a second test for the detection ofexopeptidases (enzyme).

Once again a peptide is prepared. This time the peptide contains atleast one group that contains a quencher and a fluorophore on differentamino acids. Once again the amino acid tryptophan is used as thequencher. A fluorescent dye is again used as the fluorophore. Afluorescent dye is chosen that can be coupled covalently to peptides orproteins. Furthermore, the fluorescence of the dye can be extinguishedby tryptophan.

The sequence of the peptide must be selected in such a way that thequencher and the fluorophore are spatially sufficiently close together,so long as the amino acids carrying the quencher or the fluorescent dyehave not yet been separated by the enzyme, to ensure the possibility ofan at least partial extinction of the emission of the fluorophore.Moreover, after separation of the amino acids carrying the quencher orthe fluorescent dye by the enzyme, a sufficient spatial distance must beprovided between any other quencher and the fluorophore, to achieve anat least partial increase in emission of the fluorophore.

The peptide prepared in this way is mixed in a solution with the enzymeto be detected. The intensity of the emission of the fluorophore in thesolution is determined.

In many aspects this test corresponds to that already described fordetecting an endopeptidase. It is, however, easier to detect anexopeptidase.

The exopeptidase digests the protein or peptide from its end. Itdetaches one amino acid after another. A tryptophan is also detached,among others. The dye coupled to an amino acid in the vicinity istherefore no longer extinguished, and there is an increase influorescence. The same applies when the amino acid is detached firstwith the dye.

If the substrate contains a multiplicity of tryptophan-dye groups, anincrease in fluorescence can be observed repeatedly. The effect is thena multiple increase in fluorescence.

Once again, the aforementioned red dyes can be used advantageously.

The aim is finally achieved by a third test for the detection ofendopeptidases (enzyme). This test again has many similarities and somedecisive differences.

First a peptide is prepared. A group that prevents an exopeptidase fromdigesting the peptide is arranged at one end of the peptide. The groupat the end of the peptidase that prevents the digestion of the peptideby an exopeptidase is also called a stop group or stop molecule. Thiscan be, for example, D-amino acids, DNA or PNA, which are not cleaved bythe exopeptidase.

Another possibility for providing a stop group is for an amino acid oramino acid sequence that is not cleaved by the exopeptidase to bearranged at the end of the peptide, by an appropriate choice of peptidesequence. The carboxypeptidase A as exopeptidase, for example, does notcleave beyond the amino acid proline at the end of a peptide. Thearrangement of an ordinary amino acid as stop group facilitatessynthesis of the peptide.

The exopeptidase can thus be present in solution with the peptide orprotein, without the latter being attacked.

In addition the peptide contains an identification sequence of theendopeptidase that is to be detected and can therefore serve assubstrate for the enzyme, yielding a cleavage site between two aminoacids in the identification sequence in the peptide.

Viewed from the stop group in the sequence of the peptide on the otherside of the cleavage site, the peptide contains at least one group thatcontains a quencher and a fluorophore on different amino acids.

The peptide sequence is chosen such that spatially the quencher andfluorophore are sufficiently close together, so long as the amino acidscarrying the quencher and the fluorescent dye have not yet beenseparated by an exopeptidase, to ensure the possibility of an at leastpartial extinction of the emission of the fluorophore.

Moreover, it is necessary to ensure that after separation of the aminoacids carrying the quencher and the fluorescent dye by an exopeptidase,a sufficient spatial distance is provided between any other quencher andthe fluorophore, to achieve an at least partial increase in emission ofthe fluorophore.

Finally, the peptide thus prepared, the at least one exopeptidase andthe endopeptidase that is to be detected are mixed in a solution. Thisresults in binding between peptide as substrate and endopeptidase asenzyme. The peptide is cleaved at the cleavage site within the molecule.As a result the protective stop group at the end of the substrate isseparated from the rest of the substrate. The residue that remains thenno longer has a protective stop group, and an exopeptidase can digestthe rest of the substrate.

Such an exopeptidase can be added to the solution in sufficientquantity, together with the substrate. It can thus be used as a kittogether with the substrate.

By digestion by means of the exopeptidase, the quencher and fluorophoreare separated. There is again an increase in fluorescence.

Amino acid sequences of any length can be present between the stop groupand the region of the peptide or protein carrying thequencher-and-fluorophore groups, without impairing the sensitivity ofdetection. It is thus also possible to detect enzymes that possess along identification sequence, such as HIV protease.

If a carboxypeptidase is used as exopeptidase, the stop group is placedat the C-terminal end of the peptide, so that subsequent digestion bythe carboxypeptidase can take place. If, however, an aminopeptidase isused as exopeptidase, the stop group is placed at the N-terminal end ofthe peptide.

If the substrate contains a multiplicity of quencher-and-fluorophoregroups, a multiple increase in fluorescence can again be observed. Themeasurement time can be shortened considerably as a result. There is atthe same time a marked increase in the signal. The great advantage ofmultiple marking is that with a single cleavage and thus with a singleendopeptidase to be detected, not only one, but many fluorophores areactivated. As a result we obtain a more definite increase in signal in amuch shorter time. This means that the increase in signal can even bemeasured with less sensitive instruments. There is also a shift of thelimit of detection to lower values.

This test also detects endopeptidases specifically, with the otheradvantages that have already been mentioned.

Various dyes that are bound covalently to an amino acid can be selectedas the fluorophore. A quencher can also be bound covalently to an aminoacid. A suitable combination is for example the quencher DABCYL orguanosine and as fluorophore, for example, the dyes fluorescein,coumarin or eosins.

For this third test, once again the amino acid tryptophan can be used asquencher, together with a fluorescent dye as fluorophore. It must bepossible for the fluorescent dye to couple covalently to peptides orproteins, and for its fluorescence to be extinguished by tryptophan. Theadvantages of this choice have already been explained. Once again it ispossible to use red dyes.

Among other things, for this third test there are yet otherpossibilities for producing the quencher/fluorophore pair. The quenchercan be at least one first fluorescent dye and the fluorophore at leastone second fluorescent dye, provided they can both be coupled covalentlyto peptides or proteins. What is important is that the two dyes canextinguish one another when they are spatially close together. This canresult from dimer formation or other mechanisms, such as energytransfer. After removal of the stop group by the endopeptidase that isto be detected, the exopeptidase digests the rest of the peptide chain,so that the dyes become farther apart and quenching is thus no longerpossible. The resulting increase in intensity of fluorescence ismeasured.

Preferably all the aforementioned rhodamines and oxazines are suitableas dyes for this, and in addition cyanine derivatives (e.g. Cy 3, Cy 5,Cy 7, obtainable from Amersham Biosciences), all BODIPY dyes, allcoumarins, fluorescein and Texas Red.

BRIEF DESCRIPTION OF THE DRAWING

The invention is explained in more detail below on the basis of examplesof application, shown schematically in the diagrams. Reference numbersin the individual diagrams denote the same items.

FIG. 1 is a schematic representation of the basic principle of thedetection reaction;

FIG. 2 is a schematic representation of the processes in photo-inducedelectron transfer;

FIG. 3A-C is a schematic representation of the basic principle of avariant of the detection reaction;

FIG. 4A-C is a schematic representation of the detection reaction asclaimed in FIG. 3 with multiple marking; and

FIG. 5 shows the structural formulae of the dyes mentioned.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

First Test

Detection of an Endopeptidase

FIG. 1 is a schematic representation of the principle of detection of anendopeptidase, which is represented in FIG. 1 by the scissors symbol.The peptide, consisting of four amino acids (AS) and tryptophan (Trp),serves as substrate.

A dye molecule (Dye), preferably MR 121, is coupled to the second aminoacid from the left. Possibilities for coupling are described in[Bodanszky M.: “Peptide Chemistry: A Practical Textbook”, SpringerVerlag 1986] [Anderson G. W., Zimmerman J. E., Callahlan F. M.: “The useof esters of N-Hydroxysuccinimide in Peptide Synthesis”, J. Am. Chem.Soc. 1964, 86, 1839-1842].

If, for example, we would like to detect the endopeptidase trypsin,which hydrolyzes the peptide bond between two arginines, the followingpeptide can be synthesized as substrate: Gln-Lys(MR 121)-Arg-Arg-Trp SEQID NO: 4, as shown schematically in FIG. 1.

Tryptophan acts as quencher on the dye. The mechanism of quenching isstatic quenching, in contrast to a dynamic impact quenching. The dyeforms a complex with tryptophan, called a ground-state complex, whichhardly fluoresces. In this ground-state complex there is electrontransfer from tryptophan to the excited dye molecule.

Electron transfer will be explained briefly in the following, referringto FIG. 2. This depicts fluorescence quenching of the excited dyemolecule F* by a tryptophan residue W. The black circles representelectrons. The HOMO (highest occupied molecular orbital) and the LUMO(lowest unoccupied molecular orbital) are shown in each case. The HOMOis the energetically highest molecular orbital occupied in theelectronic ground state. The LUMO is the energetically lowest molecularorbital, unoccupied in the electronic ground state; it is as a rule themolecular orbital that is occupied in the first excited state.

Basically there are two possibilities for fluorescence quenching byphoto-induced electron transfer. In the case shown on the left in FIG.2, the tryptophan residue W acts as an electron donor (Donor). Followingexcitation of the fluorophore F*, an electron is transferred from thedoubly occupied HOMO of the tryptophan residue W to the now singlyoccupied HOMO of the fluorophore F* (path 1). There is reduction of theexcited fluorophore F* by the tryptophan residue W. The electron in theLUMO of fluorophore F* can then be transferred to the now singlyoccupied HOMO of tryptophan residue W (path 2). This case occurs betweenred dyes like MR 121 and tryptophan.

In the case shown on the right in FIG. 2, the tryptophan residue W actsas an electron acceptor (Acceptor). The electron migrates from thesingly occupied LUMO of the excited fluorophore F* to the unoccupiedLUMO of tryptophan residue W (path 3). There is oxidation of the excitedfluorophore F* by the tryptophan residue W. The electron in the LUMO ofthe tryptophan residue W can then return to the HOMO of the fluorophore(path 4).

In both cases, after electron transfer the electron can no longer returnto the HOMO from the LUMO of the excited fluorophore F* by emitting aphoton. The first excited state has been deactivated without radiation.The fluorescence is quenched.

With the red dyes under examination, tryptophan always acts as anelectron donor. This knowledge makes it possible, for example, to choosesuitable dyes on the basis of their electrochemically determinedpotentials.

The fluorescence signals are preferably detected in solution in acuvette in conventional fluorescence spectrometers. This is generallycalled “homogeneous assay”. More sensitive measurements can be carriedout using confocal spectroscopy.

Instead of the fluorescence intensity, it is also possible to use thefluorescence lifetime for detection. The fluorescence lifetime isshorter in the quenched state than in the free, unquenched state.

Another possibility for detecting a quenched or unquenched state ismeasurement of changes in polarization. The polarization properties ofemissions, e.g. fluorescence signals, can vary between a quenched and anunquenched state.

In the second and third test it is also possible to use FRET systems,i.e. pairs of dyes that extinguish each other by energy transfer. Theseoften show different wavelengths in the quenched and unquenched state,which can be used for detection.

As excitation light sources it is preferable to use diode lasers,otherwise any other lasers or lamps with suitable wavelength. It ispreferable to use pulsed diode lasers for determining the extinctiontime of the fluorophores.

As well as the so-called homogeneous assay, there is also a so-calledheterogeneous assay. This generally denotes detection on a surface. Thedye-marked peptides or proteins can be bound covalently to a surface ata C-end, an N-end or via amino acid residues (e.g. cysteine or lysine).Possible surfaces are for example modified glass surfaces (accordinglychips or biochips) or (possibly magnetic) beats. For this purpose thesurfaces can be coated e.g. with linear and/or crosslinkedpolyethyleneglycols or hyaluronic acid, to prevent adsorption of themolecules.

If an amino acid, by which the peptide is bound to the surface, islocated on the same side of the cleavage site as the dye, cleavagethrough the enzyme that is to be detected has the effect that the dyebound covalently to the surface stays back and its fluorescence can nolonger be extinguished there. The fluorescence can then be detectedevanescently or by means of surface screenings.

It is also possible to immobilize the peptide on the surface in such away that the enzyme actually disrupts the bond between dye and surfaceas a result of the cleavage. The fluorescence of the unquenched dye canthen be detected in the solution.

Second Test

Detection of an Exopeptidase

The second test described at the beginning for detecting an exopeptidaseis carried out entirely similarly to the first test. The differenceswere described at the beginning.

Third Test

Detection of an Endopeptidase

FIG. 3 is a schematic representation of the principle of detection of anendopeptidase as claimed in the third test. The endopeptidase to bedetected is represented in FIG. 3A by the scissors symbol 10. Thepeptide, consisting of a stop group (Stop), two amino acids (AS),tryptophan (Trp) and another amino acid (AS), to which a dye molecule(Dye) is coupled, preferably MR 121, serves as substrate.

The endopeptidase to be detected 10 cleaves the peptide between the twoadjacent amino acids AS. This splits off the stop group (Stop) togetherwith an amino acid AS (see FIG. 3B). This makes it possible for theexopeptidase 12, which is also present in the solution and has up tillnow been prevented from digestion by the stop group (Stop), to digestthe residual peptide.

The exopeptidase 12 successively splits off first an amino acid AS andthen tryptophan (Trp). As a result of this, the dye (Dye) on the aminoacid that remains (AS) is no longer located spatially close to thetryptophan, because the tryptophan diffuses away in the solution.Therefore the dye is no longer extinguished by tryptophan. Itsfluorescence can be detected as a signal increase in the spectrometer.

For detecting HIV protease, the following peptide can be used: (Nterminus) Lys(MR 121)-Trp-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Pro-Pro (Cterminus) SEQ ID NO: 5. This peptide contains the identificationsequence for cleavage by HIV protease. This peptide can be used incombination with carboxypeptidase A, which does not cleave beyondproline. Therefore the two proline residues at the C terminus of theabove peptide serve as a stop group. HIV protease cleaves betweentyrosine and proline. Two fragments are produced: Lys(MR121)-Trp-Ser-Gln-Asn-Tyr-OH SEQ ID NO: 6 and Pro-Ile-Val-Gln-Pro-Pro SEQID NO: 7. The stop group is removed thereby and carboxypeptidase A candigest the first fragment counting from the C terminus. This alsoseparates the dye-marked lysine and tryptophan. An increase influorescence can be detected.

FIG. 4 shows the third test with signal intensification by multiplemarking.

FIG. 4A shows the peptide, as in FIG. 3A, but with an alternatingsuccession of tryptophan (Trp) and a dye-marked amino acid (AS+Dye). Ifthe endopeptidase to be detected 10 splits off the stop group (Stop)(see FIG. 4B), the exopeptidase 12 can digest the peptide successively.In the process, tryptophan and free amino acid with the bound dye(AS+Dye) are released successively (see FIG. 4C). Thus, with onecleavage of the endopeptidase, a large number of dyes is released, whoseemission is no longer extinguished. There is accordingly a rapid andstrong signal increase. This shortens the measurement time, even forhighly sensitive measurements, to minutes or seconds.

Finally, FIG. 5 shows the structure of the preferred dyes used, asmentioned at the beginning.

1. A peptide comprising: an amino acid sequence comprising at least afluorophore and tryptophan for quenching fluorescence emitted from thefluorophore, the fluorophore is not located on said tryptophan; whereinthe fluorophore is a fluorescent dye, which when covalently coupled tothe peptide emits fluorescence that is quenched by said tryptophan;wherein the fluorescent dye is an oxazine derivative or a rhodaminederivative, which absorbs and emits in a red region of visible light;wherein the fluorophore and said tryptophan are in such spatialproximity that as long as the tryptophan and the amino acid carrying thefluorescent dye have not been separated by an exopeptidase, quenching ofthe fluorescence of the fluorophore is realized; and wherein after thetryptophan and the amino acid carrying the fluorescent dye are separatedby the exopeptidase, an increase in fluorescent emission of thefluorophore is realized.
 2. A method for the detection of anexopeptidase comprising the following steps: mixing a peptide as claimedin claim 1 and the enzyme to be detected in a solution; and determiningthe intensity of emission of the fluorophore in the solution.
 3. Adevice on which a peptide as claimed in claim 1 is immobilized.
 4. Adevice having a surface on which a peptide is immobilized, said peptidecomprising: an amino acid sequence comprising at least a fluorophore andtryptophan for quenching fluorescence emitted from the fluorophore, thefluorophore is not located on said tryptophan; wherein the fluorophoreis a fluorescent dye, which when covalently coupled to the peptide emitsfluorescence that is quenched by said tryptophan; wherein thefluorescent dye is an oxazine derivative or a rhodamine derivative,which absorbs and emits in a red region of visible light; wherein thefluorophore and said tryptophan are in such spatial proximity that aslong as the tryptophan and the amino acid carrying the fluorescent dyehave not been separated by an exopeptidase, quenching of the emittedfluorescence of the fluorophore is realized; and wherein after thetryptophan and the amino acid carrying the fluorescent dye are separatedby the exopeptidase, an increase in fluorescent emission of thefluorophore is realized; and wherein after cleavage by the exopeptidaseto be detected, the fluorophore remains immobilized on the surface,whereas the quencher is no longer immobilized on the surface.
 5. Thedevice of claim 4, wherein the device is selected from the groupconsisting of glass beads, chips, biochips and magnetic beads; andwherein the surface the device is modified by layers of linear ornetworked polyethylene glycol or hyaluronic acid.